US11771752B2 - Composition for oral or nasal delivery of tetanus, diphtheria, and pertussis vaccine alone or in combination using neurotoxin associated proteins - Google Patents

Composition for oral or nasal delivery of tetanus, diphtheria, and pertussis vaccine alone or in combination using neurotoxin associated proteins Download PDF

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US11771752B2
US11771752B2 US15/064,651 US201615064651A US11771752B2 US 11771752 B2 US11771752 B2 US 11771752B2 US 201615064651 A US201615064651 A US 201615064651A US 11771752 B2 US11771752 B2 US 11771752B2
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Bal Ram Singh
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Definitions

  • This invention relates to a composition vaccine in combination with neurotoxin associated proteins for oral or nasal delivery of the vaccine. Moreover, the present invention particularly relates to oral delivery of tetanus vaccine with the help of proteins known as neurotoxin associated proteins (NAPs) from Clostridium botulinum .
  • NAPs neurotoxin associated proteins
  • the NAPs combine with vaccine candidates made of domains of tetanus neurotoxin or detoxified recombinant tetanus neurotoxin (DrTeNT) for the delivery of the tetanus vaccines by oral or nasal passage.
  • Botulinum neurotoxins is one of the most toxic substance known to man, is produced by Clostridium botulinum .
  • the clostridial neurotoxin family comprises seven BoNT serotypes (A-G), produced mainly by as a complex with a group of neurotoxin-associated proteins (NAPs).
  • the botulinum neurotoxin complex is the only known example of a protein complex where a group of proteins (NAPs) protect another protein (BoNT) against acidity and proteases of the GI tract.
  • the clostridial neurotoxin family comprises seven BoNT serotypes (A-G), produced mainly by Clostridium botulinum and the tetanus neurotoxin (TeNT), produced by Clostridium tetani .
  • A-G BoNT serotypes
  • TeNT tetanus neurotoxin
  • BoNTs and TeNT function via a similar initial physiological mechanism of action, producing paralysis by inhibition of neurotransmission.
  • BoNTs are synthesized as a single polypeptide chain comprising several domains with distinct functions that contribute to the mechanism of toxicity.
  • Other proteins produced from Clostridium botulinum form a complex with BoNT that may contribute to toxicity and the stability of the BoNT in the natural environment of food poisoning. These proteins are known as Neurotoxin Associated Proteins (NAPs).
  • NAPs Neurotoxin Associated Proteins
  • BoNT When pure BoNT is exposed to the digestive conditions with acidic fluid and proteases, BoNT degrade into inactive small peptides. Thus the pure BoNT exerts no oral toxicity. The toxicity exerts only when the BoNT is associated with NAPs, which protect BoNT against acidity and proteases of the GI tract. The NAPs not only protect BoNTs from the acidity and proteases of the GI tract but also assist in translocation of BoNT across the gut wall.
  • Tetanus is often a fatal disease caused by tetanus neurotoxin (TeNT) produced by Clostridium tetani , same family of Clostridium botulinum , which can infect wounds resulting from general cuts, needle use, unhygienic birth practices. Tetanus is a disease that is entirely preventable with immunization of the population with tetanus toxoid vaccine. The tetanus toxoid vaccine is readily available at health clinics. The tetanus vaccination has become common throughout the world at least during infant ages. Later, booster shots are recommended for whole population every 10 years.
  • TeNT tetanus neurotoxin
  • tetanus immunization is needed for the world population, as this is a disease, which often turns fatal.
  • tetanus vaccine involves needle injection, which is inconvenient and frightening for many people, especially to children.
  • tetanus vaccine is an ideal system for the development of an oral or intranasal delivery system.
  • TeNT having over 35% sequence homology with BoNTs, and sharing much of the structural and functional features of BoNT, but is not a food poison due to the lack of NAPs.
  • the present invention uses NAPs of botulinum neurotoxin for oral and intranasal delivery of vaccines, specially tetanus vaccine.
  • tetanus vaccine is prepared by treating tetanus toxin with formaldehyde, trace amount of formaldehyde is left in the vaccine to prevent possible reversion. Formaldehyde causes adverse effect upon injection.
  • Formalin-inactivated Tetanus toxoid is currently used for the immunization. It is recommended to give three to four doses in the childhood and booster dose to adult every 10 years. Tetanus toxoid is available in combination with Diphtheria and Pertussis.
  • the present mode of administration of the Formalin-inactivated Tetanus toxoid is alone or in combination with Diphtheria and Pertussis as intramuscular injection.
  • local reactions consisting of pain, erythema, tenderness and induration at the injection site are common and may be associated with systemic reactions including transient fever and irritability.
  • the reactions are basically due to the formalin present in the formulation.
  • the present invention can solve the problem of harsh digestive conditions in stomach like very low pH and bile acid, and proteases by using NAPs as adjuvants to protect the recombinant protein vaccines to go through this harsh condition. Further, with the present invention, NAPs protect heavy chain of tetanus toxoid or recombinant detoxified tetanus neurotoxin from intestinal enzymes and facilitates the vaccine to evoke the immune response.
  • the present invention solves the problem of safety concerns related to the injection of vaccines by not requiring an injection.
  • the present invention solves the problem of introduction of formaldehyde.
  • the present invention will eliminate the possibility of the introduction of formaldehyde to the patient both due to the novel method of preparation of the vaccine and delivery system.
  • the present invention will solve the problems associated with the recommended administering of an injection, because the present invention is an oral/intranasal vaccination, and it will allow mass vaccination by untrained workers, or even self-vaccination.
  • present invention will solve the problem associated with the rapid release of the vaccine. Because present invention delivered as an oral or intranasal vaccine, it will have a slower release rate compared to injection.
  • the present invention will solve the above mentioned problems related to injections.
  • the present invention is an oral vaccine, where NAPs as well as the mucous in the digestion system will provide possible protection against the above mentioned safety issues.
  • NAPs in the vaccine formulation will increase the bioavailability of the vaccine when administered orally or intranasally.
  • FIG. 1 shows a diagram of botulinum neurotoxin and its associated proteins.
  • FIG. 2 shows a diagram illustrating pTeH plasmid, which is 8461 bp in length, comprising TeNT heavy chain, TeNT HC 2580 bp in length cloned in pET32a vector between BamHI and HindIII sites, fused with thioredoxin-tag (Trx-Tag) and His-tag at N-terminal.
  • FIG. 3 shows the results of purification of recombinant TeNT heavy chain through Ni 2+ -affinity column (Ni-NTA; Nickel-Nitrilotriacetic acid) chromatography.
  • FIG. 4 shows the results of binding of recombinant tetanus heavy chain (HC) preparation with NAPs as analyzed on Ni 2+ -affinity column chromatography.
  • FIG. 5 A- 5 C shows Nucleotide sequence of DrTeNT (3951 bp, including stop codon), which encodes for the full length double mutant tetanus gene. This sequence is submitted as Sequence ID No.3
  • FIG. 6 A- 6 B shows Amino acid sequence alignment of DrTeNT (This sequence is submitted as Sequence ID No.1), reference (data from wildtype genomic DNA) this sequence is submitted as Sequence ID No.2, and CN3911 from Genebank.
  • FIG. 7 shows a schematic representation of pDrT vector map containing DrTeNT gene, shown in grey color (3966 bp include 6-His tag).
  • FIG. 8 A- 8 B shows Dr TeNT protein purification process and the end product.
  • 8 A shows Dr TeNT was purified by using NTA column.
  • Dr TeNT has his-tag at C-terminal and the molecular weight of Dr TeNT is about 150 kDa, and the protein eluted from column around 100 mm-200 mM Imidazole.
  • 8 B shows the purified Dr TeNT.
  • FIG. 9 Binding of NAPs and DrTeNT is demonstrated using a G-100 gel filtration column chromatography performed in 50 mM sodium phosphate buffer, pH 5.8, containing 200 mM NaCl, showing a single peak elution of the mixture containing all the NAPs and DrTeNT.
  • FIG. 10 SDS-PAGE analysis of DrTeNT, NAPs, DrTeNT+NAPs load to the G-100 column, and DrTeNT+NAPs elution pool from the G-100 gel filtration column.
  • FIG. 11 Schematic model of the DrTeNT-NAPs complex.
  • FIG. 12 The immune response of the proposed construct with oral delivery as well as subcutaneous delivery.
  • Main object of the present invention is to provide a novel composition for the delivery of vaccine by oral or nasal administration.
  • Another object of present invention is to provide a novel composition for oral or nasal delivery of tetanus vaccine.
  • Yet another objective of present invention is to provide a novel composition for oral or nasal delivery of Tetanus, Diphtheria, and Pertussis Vaccine either alone or in combination thereof.
  • Another object of present invention is the use of neurotoxin associated protein (NAPs) for the oral or nasal delivery of the vaccine.
  • NAPs neurotoxin associated protein
  • Another object of the present invention is the use of neurotoxin associated protein (NAPs) of Clostridium botulinum.
  • NAPs neurotoxin associated protein
  • Yet another object of present invention is to provide a process for preparation of NAPs from type A Clostridium botulinum.
  • Yet another objective of present invention is to provide a process for the preparation of tetanus toxoid vaccine.
  • Yet another object of the present invention is to provide a process for the preparation of tetanus toxoid vaccine.
  • Yet another object of present invention is to provide first and second generation process for the preparation of tetanus toxoid vaccine.
  • Yet another object of present invention is to provide a process for the third generation tetanus vaccine as Detoxified recombinant tetanus neurotoxin (DrTeNT).
  • DrTeNT amino acid sequence is submitted as Sequence ID No.1
  • Yet another object of present invention is to provide a testing model for the effectiveness of the oral and intranasal delivery of the vaccine.
  • Yet another object of present invention is to provide a testing model in rabbit or another suitable model for the effectiveness of the oral and intranasal delivery of the vaccine.
  • Another object of the present invention is oral or nasal delivery of vaccine.
  • Yet another object of the present invention is oral or nasal delivery of Tetanus, Botulinum, Diphtheria, and Pertussis Vaccine either alone or in combination thereof.
  • Another object of the present invention is to provide process for preparation of composition for the vaccine.
  • present invention provides a composition of neurotoxin associated proteins and vaccine.
  • neurotoxin associated proteins is of botulinum neurotoxin.
  • NAPs neurotoxin associated proteins
  • Another embodiment of the present invention wherein the vaccine selected from the group consisting of tetanus, diphtheria and pertussis alone or in combination thereof.
  • tetanus vaccine is non-toxic protein domain of TeNT.
  • the non-toxic protein domain of TeNT is selected from heavy (H) chain or detoxified recombinant TeNT (DrTeNT) alone or in combination thereof.
  • DrTeNT detoxified recombinant TeNT
  • vaccine is made of a fusion with DrTeNT or any of its derived fragments with diphtheria vaccine element.
  • vaccine DrTeNT is combined with pertussis vaccine element as a fusion protein.
  • Another embodiment of the present invention wherein vaccine DrTeNT combined with vaccine elements of diphtheria and pertussis vaccine elements as a fusion protein.
  • FIG. 1 For embodiment of the present invention, is a process for the preparation of second generation tetanus toxoid vaccine, ie, tetanus heavy chain comprising steps of;
  • the present invention employs use of neurotoxin associated proteins (NAPs) of botulinum neurotoxin (BoNT) produced by Clostridium botulinum as a delivery vehicles of tetanus vaccine.
  • BoNT is a food poison produced in the form of a complex with NAPs, which protect it from the low pH and proteases of the gastro-intestinal tract.
  • NAPs are known to help in translocation of the BoNT across the mucosal layer of intestine as well as the nasal passage. Given the similarity of structure of BoNT and TeNT and their common mode of action, NAPs bind and protect and translocate a tetanus vaccine across the mucosal layer.
  • Clostridium botulinum produces seven serotypes of botulinum neurotoxins (A-G). It is basically made of two protein chains one is 100 kDa polypeptide heavy chain and another is 50 kDa polypeptide light chain bridged through disulphide link.
  • Botulinum neurotoxins is synthesized in a complex form, in which neurotoxin is surrounded by several non-toxin proteins known as neurotoxin associated proteins (NAPs). NAPs have been shown to have two major roles in the intoxication process of botulism. The first role is the assistance of NAPs in the translocation of the BoNT across the intestinal mucosal layer. The second role is NAPs protect the BoNT against acidity and proteolytic attack of the enzymes of gastric juice.
  • the TeNT having over 35% sequence homology with BoNTs, and sharing much of the structural and functional features of BoNT, but is not a food poison due to the lack of NAPs.
  • NAPs from one serotype of BoNT bind and protect another serotype of BoNT, thus these can also bind, protect and deliver vaccines, specially TeNT or TeNT vaccine due to sequence homology and common structural and functional domains. Therefore, the present invention uses BoNT's NAPs for oral and intranasal delivery of vaccines, specially tetanus vaccine.
  • Botulinum and tetanus neurotoxins have several common features including over 35% sequence homology, and antagonistic effects of heterologous protein fragments. Both the neurotoxins have similar secondary structural contents, and both have their ‘catalytic domain’ light chain on the C-terminal of their amino acid sequence. Tetanus is capable of causing botulism symptoms at high concentrations and blocks the release of acetylcholine from the presynaptic membranes just like the BoNT.
  • BoNT is a food poison produced in the form of a complex with NAPs (see FIG. 1 ).
  • NAPs protect BoNT from the low pH and proteases of the gastro-intestinal tract (Mahmut et al., 2002).
  • NAPs are known to help translocate the BoNT across the mucosal layer of intestine as well as the nasal passage (Fujinaga et al., 2004).
  • Hn-33 Given the similarity of structure of BoNT and TeNT and their common mode of action, Hn-33 is likely to bind and protect and translocate a tetanus vaccine across the mucosal layer (Mahmut et al., 2002).
  • Tetanus toxoid vaccine a second generation vaccine is prepared based on the non-toxic protein domain of TeNT, such as the heavy (H) chain for demonstrating its binding and delivery by NAPs.
  • the first steps include the preparation of a second generation tetanus vaccine based on the non-toxic heavy chain of the TeNT.
  • FIG. 2 shows the schematic genomic map of pET32a, showing the location of TeNT heavy chain TeNT heavy chain plasmids were transferred to BL21(DE3) competent cells, and grown in the LB media.
  • Second step is the preparation of NAPs from type A Clostridium botulinum
  • Present invention also provides method of preparation of the clone of DrTeNT as the third generation tetanus vaccine.
  • the third generation tetanus vaccine is prepared from and/or detoxified recombinant TeNT (DrTeNT) is cloned in pET32a vector between BamHI and HindIII sites with the N-terminal his-tag.
  • Botulinum neurotoxin and purification of NAPs is done as per known method in the art (Kukreja et al., Toxicon, 2009 (53) 616-624).
  • the cell paste culture suspended in about 1 L, 25 mM phosphate buffer, pH 7.4, containing 200 mM sodium chloride and 1% of TRITON®-X-100 added in the buffer (called basic buffer).
  • basic buffer a buffer for sonication, the cell lysate was centrifuged at 12,000 rpm for about 1 hour by using Thermo Scientific® Sorvall® Legend® RT Centrifuge and FIBERLite® F15-8x50C rotor, and then the supernatant is poured into a clean tube. The extract is thus obtained and is loaded to the pre-equilibrated Ni-NTA column.
  • the detoxified recombinant TeNT (DrTeNT) was prepared by cloning the TeNT gene and mutating two active site glutamic amino acid residues, E234 and E271 each to alanine residues. Cloning of Dr TeNT involved three steps: (1) pBN3 vector was used for cloning TeTx Light chain. (2) Two active sites mutation, E234A and E271A, was done which corresponds to E224 and E262 of BoNT/A active sites. (3) HC portion of TeNT was fused to construct full length tetanus neurotoxin plasmid with two active sites mutation, E234A and E271A.
  • E. coli BL21 (DE3) was used for the protein expression vector. After induction there were a large band at 150 kDa position which was the size of full length recombinant tetanus neurotoxin. After Ni-NTA affinity column, about 5 mg/ml pure protein was obtained which was soluble and stable.
  • FIG. 3 shows the results of purification of recombinant TeNT heavy chain through Ni 2+ -affinity column chromatography.
  • Lane1 is MW standards.
  • Lane 2 is Ni-NTA (Nickel-Nitrilotriacetic acid) affinity column washing with 10 mM imidazole added in the basic buffer.
  • Lanes 3-6 are elution with 50 mM imidazole concentration.
  • Lane 7 is elution with 100 mM Imidazole.
  • Lane 8 is elution with 200 mM imidazole.
  • tetanus heavy chain has N-terminal his-tag which can bind to Ni-NTA affinity column, but NAPs which is purified from botulinum neurotoxin complex cannot bind to the Ni-NTA affinity column, so pull down assay is used for analysis to demonstrate if heavy chain would bind with NAPs. If Heavy chain has interaction with NAPs, both HC and NAPs would remain bound to NTA column and will be released together from Ni-NTA affinity column after eluting with imidazole.
  • tetanus heavy chain and NAPs are mixed together at pH 5.8. Binding reaction is carried out at room temperature for 2 hours. 25 mM phosphate buffer, pH 5.8, containing 200 mM NaCl is used for procedure of pull down assay. After reaction, the reaction mixture is diluted to a final imidazole concentration lower than 5 mM. The reaction mixture is loaded to the Ni-NTA affinity column, and 10 mM imidazole is added to the basic buffer to wash column at 20 ⁇ bed volume, then the protein is eluted stepwise with 50 mM, 100 mM, 200 mM, 500 mM imidazole. The eluents are examined with SDS-PAGE to check the purity and intactness of the TeNT heavy chain, as illustrated in FIG. 3 .
  • Tetanus heavy chain purified from different batches is used for the binding experiments. After HC and NAPs reaction at room temperature, reaction mixture is reloaded to the Ni-NTA affinity column, with 10 mM Imidazole added to the basic phosphate buffer for washing column. After then protein is eluted with 50 mM, 100 mM, 200 mM imidazole. From the gel, it was observed that Tetanus heavy chain and NAPs were eluted together at 50 mM imidazole concentration ( FIG. 4 ).
  • FIG. 4 shows binding of recombinant tetanus heavy chain (HC) preparation with NAPs as analyzed on Ni 2+ -affinity column chromatography.
  • Lane 1 is MW.
  • Lane 2 is intact heavy chain protein.
  • Lane 3 is intact NAPs.
  • Lane 4 is elution from Ni-NTA affinity column with imidazole 50 mM after loading HC and NAPs reaction mixture to Ni-NTA affinity column, Lane 4 showed HC and NAPs came together at 50 mM imidazole concentration.
  • NAPs results shown in FIG. 4 , lane 4, demonstrating that the NAPs successfully bound to the tetanus heavy chain. This observation would suggest that the tetanus heavy chain could be carried through the digestive system when administered orally in combination with NAPs.
  • the NAPs are also expected to deliver the tetanus heavy chain or whole tetanus toxin across the intestinal mucosal layer.
  • FIG. 5 shows nucleotide sequence of DrTeNT (3951 bp, including stop codon) which encodes for the full length double mutant tetanus gene.
  • the start codon is enclosed in a green box, which is the same start for TeNT light chain; the double mutation sites are enclosed in orange boxes, GAA (Glu) to GCA (ALA) mutation; the heavy chain start codon (TCA) is enclosed in black box; and the stop codon is enclosed in a red box following the six His tag.
  • FIG. 6 shows the amino acid sequence alignment of DrTeNT, reference (data from wildtype genomic DNA), and CN3911 from Genebank.
  • the two red boxes show that two active sites which got mutated in DrTeNT from wild type.
  • the black boxes show that amino acid sequence variation from GeneBank C. tetani strain.
  • FIG. 7 shows a schematic representation of pDrT vector map containing DrTeNT gene (3966 bp include 6-His tag). DrTeNT was cloned in pBN3 vector (3452 bp), between E.coRI and PstI sites. The new construction of pDrT vector is 7418 bp in length.
  • DrTeNT gene in E. coli has been achieved and better than 99% pure DrTeNT is purified on a Ni-NTA affinity column ( FIG. 8 ).
  • Binding of NAPs and DrTeNT is demonstrated using a G-100 gel filtration column chromatography performed in 50 mM sodium phosphate buffer, pH 5.8, containing 200 mM NaCl, showing a single peak elution of the mixture containing all the NAPs and DrTeNT ( FIG. 9 ). Binding of NAPs and DrTeNT is confirmed by SDS-PAGE gel ( FIG. 10 ). A model of the DrTeNT and NAPs complex is shown in FIG. 11 .
  • the binding of the NAPs to tetanus heavy chain or DrTeNT is done using high-performance liquid chromatography (HPLC).
  • HPLC high-performance liquid chromatography
  • both NAPs and HC (used as a control) run separately when passed through the size exclusion column. After mixing them together at low pH conditions for binding with each other, and the mixture will appear as a single elution band.
  • the binding of the NAPs to tetanus heavy chain is done using enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the tetanus heavy chain is coated on the 96 well plate at various different concentration
  • Incubation of NAPs labeled with Horseradish Peroxidase (HRP) will show concentration dependent binding as monitored by colorimetric method by adding HRP substrate.
  • the binding of the NAPs to tetanus heavy chain or DrTeNT is done using a BIACORE® surface plasmon resonance. In this preferred embodiment binding is evaluated on a gold chip.
  • the binding of the NAPs to tetanus heavy chain or DrTeNT is done using isothermal calorimetry.
  • titration of a given concentration of NAPs with varying concentrations of tetanus vaccine candidate will show changes in heat evolved, thus allowing calculation of binding constant.
  • the binding of the NAPs to tetanus heavy chain or DrTeNT is done using Fluorescence anisotropy.
  • the tetanus vaccine candidate can be labelled with a fluorescent probe, and titrated with the NAPs by monitoring fluorescence anisotropy.
  • the binding of the NAPs to tetanus heavy chain, DrTeNT, or any other fragment thereof is demonstrated with x-ray crystallography.
  • the binding of the NAPs to tetanus heavy chain or DrTeNT is demonstrated with electron microscopy.
  • the binding of the NAPs to tetanus heavy chain, DrTeNT, or any other fragment thereof is demonstrated with NMR spectroscopy.
  • the binding of the NAPs to tetanus heavy chain, DrTeNT, or any other fragment thereof is demonstrated with molecular dynamic simulations.
  • the binding of the NAPs to tetanus heavy chain, DrTeNT, or any other fragment thereof is demonstrated with circular dichroism spectroscopy.
  • the binding of the NAPs to tetanus heavy chain, DrTeNT, or any other fragment thereof is demonstrated with FTIR spectroscopy.
  • the effectiveness of inducing an immune response with oral delivery as well as subcutaneous delivery is shown in FIG. 12 .
  • the first bar Sub Q Mix is subcutaneous mixture of NAPs with DrTeNT, next bar is same material via oral root and then oral DrTeNT alone. Although subcutaneous is more effective to illicit a response it seem that via oral mixture of both articles are more effective than the DrTeNT alone.
  • neurotoxin associated protein from botulinum neurotoxin complex used as a delivery system for a tetanus vaccine.
  • a neurotoxin associated protein from botulinum neurotoxin used as a delivery system for a tetanus vaccine.
  • the tetanus heavy chain is used.
  • the tetanus heavy chain linked to a botulinum toxin fragment is used.
  • DrTeNT linked to DrBoNT is used.
  • a neurotoxin associated protein from botulinum neurotoxin complex is used as delivery system to botulinum vaccine.
  • neurotoxin associated proteins from botulinum neurotoxin complex is used as delivery system to botulinum vaccine.
  • DrBoNT or any other fragment thereof is used as a vaccine.
  • the neurotoxin associated protein is obtained from Clostridium botulinum.
  • the neurotoxin associated protein is obtained from recombinant protein expression system.
  • the neurotoxin associated protein is obtained from Clostridium botulinum bind to tetanus heavy chain.
  • the detoxified recombinant form of whole tetanus neurotoxin (DrTeNT) containing mutated light chain and native heavy chain is used as vaccine alone for oral and intranasal delivery.
  • DrTeNT whole tetanus neurotoxin
  • the detoxified form of whole tetanus neurotoxin containing mutated light chain and native heavy chain is used as vaccine in combination with the neurotoxin associated protein for oral and intranasal delivery.
  • DrTeNT is combined with diphtheria vaccine element as a fusion protein.
  • DrTeNT is combined with pertussis vaccine element as a fusion protein.
  • DrTeNT is combined with vaccine elements of diphtheria and pertussis vaccine elements as a fusion protein.
  • the group of neurotoxin associated proteins is used as a delivery vehicle for tetanus vaccine delivery.
  • the tetanus vaccine is heavy chain of tetanus.
  • the tetanus vaccine is light chain of tetanus.
  • the tetanus vaccine is any fragment of tetanus.
  • the vaccine is made of a fusion with DrTeNT or any of its derived fragments with diphtheria vaccine element.
  • the vaccine is made of a fusion with DrTeNT or any of its derived fragments with pertussis vaccine element.
  • the vaccine is made of a fusion with DrTeNT or any of its derived fragments with pertussis and diphtheria vaccine elements.
  • the drug is created by combining DrTeNT or any of its fragments with any other drug candidate.
  • Present invention gives a novel composition of neurotoxin associated protein and vaccine.
  • the neurotoxin associated protein helps in oral or nasal delivery of vaccine, especially tetanus vaccine with or without diphtheria and pertussis.
  • present invention gives a novel process for the preparation of second and third generation of vaccine.
  • Present invention also provides a novel process for the preparation of composition of neurotoxin associated protein and vaccine for oral delivery or nasal delivery.
  • Present invention also provides a method for evaluation of the efficiency of the oral delivery of vaccine.

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Abstract

The present invention describes a neurotoxin associated protein from botulinum neurotoxin complex used as an oral or nasal delivery system for a vaccine. The vaccine is selected from tetanus, diphtheria and pertussis alone or in combination. Further the oral or nasal delivery of tetanus vaccine in combination with other drug molecules.

Description

TECHNICAL FIELD
This invention relates to a composition vaccine in combination with neurotoxin associated proteins for oral or nasal delivery of the vaccine. Moreover, the present invention particularly relates to oral delivery of tetanus vaccine with the help of proteins known as neurotoxin associated proteins (NAPs) from Clostridium botulinum. The NAPs combine with vaccine candidates made of domains of tetanus neurotoxin or detoxified recombinant tetanus neurotoxin (DrTeNT) for the delivery of the tetanus vaccines by oral or nasal passage.
BACKGROUND OF THE INVENTION
Botulinum neurotoxins (BoNT), is one of the most toxic substance known to man, is produced by Clostridium botulinum. The clostridial neurotoxin family comprises seven BoNT serotypes (A-G), produced mainly by as a complex with a group of neurotoxin-associated proteins (NAPs). The botulinum neurotoxin complex is the only known example of a protein complex where a group of proteins (NAPs) protect another protein (BoNT) against acidity and proteases of the GI tract. The clostridial neurotoxin family comprises seven BoNT serotypes (A-G), produced mainly by Clostridium botulinum and the tetanus neurotoxin (TeNT), produced by Clostridium tetani. Although the BoNTs and TeNT function via a similar initial physiological mechanism of action, producing paralysis by inhibition of neurotransmission. BoNTs are synthesized as a single polypeptide chain comprising several domains with distinct functions that contribute to the mechanism of toxicity. Other proteins produced from Clostridium botulinum form a complex with BoNT that may contribute to toxicity and the stability of the BoNT in the natural environment of food poisoning. These proteins are known as Neurotoxin Associated Proteins (NAPs). When pure BoNT is exposed to the digestive conditions with acidic fluid and proteases, BoNT degrade into inactive small peptides. Thus the pure BoNT exerts no oral toxicity. The toxicity exerts only when the BoNT is associated with NAPs, which protect BoNT against acidity and proteases of the GI tract. The NAPs not only protect BoNTs from the acidity and proteases of the GI tract but also assist in translocation of BoNT across the gut wall.
Tetanus is often a fatal disease caused by tetanus neurotoxin (TeNT) produced by Clostridium tetani, same family of Clostridium botulinum, which can infect wounds resulting from general cuts, needle use, unhygienic birth practices. Tetanus is a disease that is entirely preventable with immunization of the population with tetanus toxoid vaccine. The tetanus toxoid vaccine is readily available at health clinics. The tetanus vaccination has become common throughout the world at least during infant ages. Later, booster shots are recommended for whole population every 10 years.
One hundred percent tetanus immunization is needed for the world population, as this is a disease, which often turns fatal. In addition, there is a need for booster shots of tetanus every 10 years, which frequently is overlooked. Current delivery system for tetanus vaccine involves needle injection, which is inconvenient and frightening for many people, especially to children. Currently there are over 200,000 annual tetanus deaths (mostly maternal and neonatal) worldwide which could be prevented with effective immunization. Therefore, tetanus vaccine is an ideal system for the development of an oral or intranasal delivery system.
There is a great need for child vaccination by developing a needle free immunization systems for a much needed tetanus vaccination. Currently most vaccines are still injected into body by needles. It is stressful and painful, especially to young children whom get most of their vaccines at early ages. Oral delivery method proposed for administration of tetanus vaccine will be less painful, and safer. There will be no issue of contaminated needles.
Further, currently many vaccines cannot be delivered by oral route because the harsh digestive conditions in stomach like very low pH and bile acid, and proteases. Moreover, currently there are several safety concerns related to the injection of vaccines, e.g., allergic reaction, and contaminated antigen.
The TeNT, having over 35% sequence homology with BoNTs, and sharing much of the structural and functional features of BoNT, but is not a food poison due to the lack of NAPs. NAPs from one serotype of BoNT binding and protecting another serotype of BoNT, therefore NAPs can also bind, protect and translocate vaccines across the gut wall, specially TeNT or TeNT vaccine due to sequence homology.
Therefore the present invention uses NAPs of botulinum neurotoxin for oral and intranasal delivery of vaccines, specially tetanus vaccine.
At present tetanus vaccine is prepared by treating tetanus toxin with formaldehyde, trace amount of formaldehyde is left in the vaccine to prevent possible reversion. Formaldehyde causes adverse effect upon injection.
Further, currently tetanus vaccination is recommended every 10 years, most of the adult population is not up to date on tetanus vaccination due to the difficulties associated with administration of the vaccination.
Formalin-inactivated Tetanus toxoid is currently used for the immunization. It is recommended to give three to four doses in the childhood and booster dose to adult every 10 years. Tetanus toxoid is available in combination with Diphtheria and Pertussis. The present mode of administration of the Formalin-inactivated Tetanus toxoid is alone or in combination with Diphtheria and Pertussis as intramuscular injection. On the intramuscular injection local reactions consisting of pain, erythema, tenderness and induration at the injection site are common and may be associated with systemic reactions including transient fever and irritability. The reactions are basically due to the formalin present in the formulation. Thus, there is a continual need to develop oral tetanus vaccine alone or in combination with diphtheria and pertussis.
Availability of an oral delivery system would facilitate the vaccination schemes for pregnant women, infants and the world population in general. Currently no such vaccine delivery system is available, nor there is any effort known to be underway in this direction.
The present invention can solve the problem of harsh digestive conditions in stomach like very low pH and bile acid, and proteases by using NAPs as adjuvants to protect the recombinant protein vaccines to go through this harsh condition. Further, with the present invention, NAPs protect heavy chain of tetanus toxoid or recombinant detoxified tetanus neurotoxin from intestinal enzymes and facilitates the vaccine to evoke the immune response.
Further, the present invention solves the problem of safety concerns related to the injection of vaccines by not requiring an injection.
Further, the present invention solves the problem of introduction of formaldehyde. The present invention will eliminate the possibility of the introduction of formaldehyde to the patient both due to the novel method of preparation of the vaccine and delivery system.
Further, the present invention will solve the problems associated with the recommended administering of an injection, because the present invention is an oral/intranasal vaccination, and it will allow mass vaccination by untrained workers, or even self-vaccination.
Further, the present invention will solve the problem associated with the rapid release of the vaccine. Because present invention delivered as an oral or intranasal vaccine, it will have a slower release rate compared to injection.
Further, the present invention will solve the above mentioned problems related to injections. The present invention is an oral vaccine, where NAPs as well as the mucous in the digestion system will provide possible protection against the above mentioned safety issues.
Further, NAPs in the vaccine formulation will increase the bioavailability of the vaccine when administered orally or intranasally.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a diagram of botulinum neurotoxin and its associated proteins.
FIG. 2 shows a diagram illustrating pTeH plasmid, which is 8461 bp in length, comprising TeNT heavy chain, TeNT HC 2580 bp in length cloned in pET32a vector between BamHI and HindIII sites, fused with thioredoxin-tag (Trx-Tag) and His-tag at N-terminal.
FIG. 3 shows the results of purification of recombinant TeNT heavy chain through Ni2+-affinity column (Ni-NTA; Nickel-Nitrilotriacetic acid) chromatography.
FIG. 4 shows the results of binding of recombinant tetanus heavy chain (HC) preparation with NAPs as analyzed on Ni2+-affinity column chromatography.
FIG. 5A-5C shows Nucleotide sequence of DrTeNT (3951 bp, including stop codon), which encodes for the full length double mutant tetanus gene. This sequence is submitted as Sequence ID No.3
FIG. 6A-6B shows Amino acid sequence alignment of DrTeNT (This sequence is submitted as Sequence ID No.1), reference (data from wildtype genomic DNA) this sequence is submitted as Sequence ID No.2, and CN3911 from Genebank.
FIG. 7 shows a schematic representation of pDrT vector map containing DrTeNT gene, shown in grey color (3966 bp include 6-His tag).
FIG. 8A-8B shows Dr TeNT protein purification process and the end product. 8A shows Dr TeNT was purified by using NTA column. Dr TeNT has his-tag at C-terminal and the molecular weight of Dr TeNT is about 150 kDa, and the protein eluted from column around 100 mm-200 mM Imidazole. 8B shows the purified Dr TeNT.
FIG. 9 . Binding of NAPs and DrTeNT is demonstrated using a G-100 gel filtration column chromatography performed in 50 mM sodium phosphate buffer, pH 5.8, containing 200 mM NaCl, showing a single peak elution of the mixture containing all the NAPs and DrTeNT.
FIG. 10 . SDS-PAGE analysis of DrTeNT, NAPs, DrTeNT+NAPs load to the G-100 column, and DrTeNT+NAPs elution pool from the G-100 gel filtration column.
FIG. 11 . Schematic model of the DrTeNT-NAPs complex.
FIG. 12 . The immune response of the proposed construct with oral delivery as well as subcutaneous delivery.
 OBJECT OF THE INVENTION
Main object of the present invention is to provide a novel composition for the delivery of vaccine by oral or nasal administration.
Another object of present invention is to provide a novel composition for oral or nasal delivery of tetanus vaccine.
Yet another objective of present invention is to provide a novel composition for oral or nasal delivery of Tetanus, Diphtheria, and Pertussis Vaccine either alone or in combination thereof.
Another object of present invention is the use of neurotoxin associated protein (NAPs) for the oral or nasal delivery of the vaccine.
Another object of the present invention is the use of neurotoxin associated protein (NAPs) of Clostridium botulinum.
Yet another object of present invention is to provide a process for preparation of NAPs from type A Clostridium botulinum.
Yet another objective of present invention is to provide a process for the preparation of tetanus toxoid vaccine.
Yet another object of the present invention is to provide a process for the preparation of tetanus toxoid vaccine.
Yet another object of present invention is to provide first and second generation process for the preparation of tetanus toxoid vaccine.
Yet another object of present invention is to provide a process for the third generation tetanus vaccine as Detoxified recombinant tetanus neurotoxin (DrTeNT). DrTeNT amino acid sequence is submitted as Sequence ID No.1
Yet another object of present invention is to provide a testing model for the effectiveness of the oral and intranasal delivery of the vaccine.
Yet another object of present invention is to provide a testing model in rabbit or another suitable model for the effectiveness of the oral and intranasal delivery of the vaccine.
Another object of the present invention is oral or nasal delivery of vaccine.
Yet another object of the present invention is oral or nasal delivery of Tetanus, Botulinum, Diphtheria, and Pertussis Vaccine either alone or in combination thereof.
Another object of the present invention is to provide process for preparation of composition for the vaccine.
BRIEF SUMMARY OF THE INVENTION
Accordingly present invention provides a composition of neurotoxin associated proteins and vaccine.
Another embodiment of the present invention wherein the neurotoxin associated proteins (NAPs) is of botulinum neurotoxin.
Another embodiment of the present invention wherein the neurotoxin associated proteins (NAPs) are used as a delivery vehicle for tetanus vaccine delivery.
Another embodiment of the present invention wherein the vaccine selected from the group consisting of tetanus, diphtheria and pertussis alone or in combination thereof.
Another embodiment of the present invention wherein tetanus vaccine is non-toxic protein domain of TeNT.
Another embodiment of the present invention the non-toxic protein domain of TeNT is selected from heavy (H) chain or detoxified recombinant TeNT (DrTeNT) alone or in combination thereof.
Another embodiment of the present invention wherein the detoxified recombinant TeNT (DrTeNT) is selected from mutated light chain or native heavy chain or combination thereof.
Another embodiment of the present invention wherein vaccine is made of a fusion with DrTeNT or any of its derived fragments with diphtheria vaccine element.
Another embodiment of the present invention wherein vaccine DrTeNT is combined with pertussis vaccine element as a fusion protein.
Another embodiment of the present invention wherein vaccine DrTeNT combined with vaccine elements of diphtheria and pertussis vaccine elements as a fusion protein.
Further embodiment of the present invention is a process for the preparation of second generation tetanus toxoid vaccine, ie, tetanus heavy chain comprising steps of;
    • a. Induction of E. coli culture OD600=0.5 by adding 0.2 mM IPTG
    • b. Growing culture for 14-16° C. at 14 to 20 hours
    • c. the cell paste suspends in 25 mM phosphate buffer containing 200 mM sodium chloride
    • d. add 1% of TRITON®-X-100 to the buffer
    • e. Sonicate for a period of 3 minutes (3-5 sec on/off pulse) at 4° C. on cold beads.
    • f. Centrifuge the culture for 60 to 90 minutes.
    • g. Supernatant is collected; and
    • h. Supernatant purified using Ni-NTA affinity column.
    • i. Combine the pool which has less contaminated bands and concentrate the pool by using CENTRIPREP®-30.
      Another embodiment of the present invention, the process, wherein the pH of buffer in the range of 7.2 to 8.0
      Another embodiment of the present invention, the process. wherein the pH of buffer is preferably 7.4
      Another embodiment of the present invention wherein, in process, rpm of centrifuge rotation is 8000-15000 rpm per hour for 45 to 90 minutes.
      Another embodiment of the present invention wherein eluent is imidazole solution.
      Yet another embodiment of the present invention wherein the concentration of imidazole solution is 10 mM, 50 mM, 100 mM, 200 mM and 500 mM.
      Accordingly the present invention relates a process for preparation of third generation detoxified recombinant tetanus toxoid vaccine comprising steps of;
    • a. pBN3 vector was used for cloning TeTx Light chain in a BL21(DE3) strain for expression of the plasmid.
    • b. Two active sites mutation, E234A and E271A, was done which corresponds to E224 and E262 of BoNT/A active sites.
    • c. HC portion of TeNT was fused to construct full length tetanus neurotoxin plasmid with two active sites mutation, E234A and E271A.
    • d. Purified the large band at 150 kDa protein position using Ni-NTA affinity column.
      Yet another embodiment of the present invention a process wherein strain used for expression of the plasmid E. coli BL21 (DE3).
      Yet another embodiment of the present invention is a process wherein purified large band at 150 kDa protein is obtained as soluble and stable.
      Accordingly present invention relates a process for the preparation of vaccine and NAPs composition comprises
    • a. mixing equimolar ratio of vaccine and NAPs at pH 5.5 to 6.2
    • b. Reacting at an ambient temperature for 2-3 hours
    • c. 25 mM phosphate buffer of pH 5.8 containing 200 mM sodium chloride solution charged to step 2
    • d. reacting above obtained reaction mixture with 10 mM imidazole solution
    • e. The reaction mixture further loaded to G-100 column
    • f. eluted the column to get the pure NAPs-vaccine composition.
      Yet another embodiment of the present invention is a process wherein vaccine is selected from the group consisting of tetanus, diphtheria, pertussis alone or combination thereof.
      Yet another embodiment of the present invention is a process, wherein temperature in range of 20-30° C.
      Yet another embodiment of the present invention is a process where in preferred pH is 5.8.
      Yet another embodiment of the present invention is a process wherein tetanus vaccine is a non-toxic protein domain of TeNT.
      Yet another embodiment of the present invention is a process wherein tetanus vaccine is selected from heavy (H) chain or detoxified recombinant TeNT (DrTeNT) alone or in combination thereof.
      Yet another embodiment of the present invention is a process wherein, detoxified recombinant TeNT is selected from mutated light chain or native heavy chain or combination thereof.
      Yet another embodiment of the present invention is a process wherein NAPs is from botulinum neurotoxin.
      Yet another embodiment of the present invention is a process wherein the NAPs are obtained from any serotype of C. botulinum.
      Yet another embodiment of the present invention is a process wherein tetanus vaccine is linked to detoxified botulinum neurotoxin (DrBoNT).
      Yet another embodiment of the present invention is a process wherein tetanus vaccine is linked to a fragment of detoxified botulinum neurotoxin.
      Yet another embodiment of the present invention is a process wherein tetanus vaccine is linked to NAPs of botulinum neurotoxin.
      Yet another embodiment of the present invention is the composition is used for the oral or nasal delivery of vaccine.
      Yet another embodiment of the present invention is the vaccine is selected from the group consisting of tetanus, diphtheria or pertussis alone or in combination thereof.
      Yet another embodiment of the present invention is the tetanus vaccine is non-toxic protein domain of TeNT.
      Yet another embodiment of the present invention is the non toxic protein domain of TeNT is selected from heavy (H) chain or detoxified recombinant TeNT (DrTeNT) alone or in combination thereof.
      Yet another embodiment of the present invention is the detoxified recombinant TeNT (DrTeNT) is selected from mutated light chain or native heavy chain or combination thereof.
      Yet another embodiment of the present invention is the vaccine is made of a fusion with DrTeNT or any of its derived fragments with diphtheria vaccine element.
      Yet another embodiment of the present invention is the DrTeNT is combined with pertussis vaccine element as a fusion protein.
      The vaccine as claimed in claim 32, wherein DrTeNT is combined with vaccine elements of diphtheria and pertussis vaccine elements as a fusion protein.
DETAILED DESCRIPTION OF THE INVENTION
The present invention employs use of neurotoxin associated proteins (NAPs) of botulinum neurotoxin (BoNT) produced by Clostridium botulinum as a delivery vehicles of tetanus vaccine. BoNT is a food poison produced in the form of a complex with NAPs, which protect it from the low pH and proteases of the gastro-intestinal tract. In addition, NAPs are known to help in translocation of the BoNT across the mucosal layer of intestine as well as the nasal passage. Given the similarity of structure of BoNT and TeNT and their common mode of action, NAPs bind and protect and translocate a tetanus vaccine across the mucosal layer.
Clostridium botulinum produces seven serotypes of botulinum neurotoxins (A-G). It is basically made of two protein chains one is 100 kDa polypeptide heavy chain and another is 50 kDa polypeptide light chain bridged through disulphide link. Botulinum neurotoxins is synthesized in a complex form, in which neurotoxin is surrounded by several non-toxin proteins known as neurotoxin associated proteins (NAPs). NAPs have been shown to have two major roles in the intoxication process of botulism. The first role is the assistance of NAPs in the translocation of the BoNT across the intestinal mucosal layer. The second role is NAPs protect the BoNT against acidity and proteolytic attack of the enzymes of gastric juice. Therefore it becomes a perfect delivery system for vaccine. The TeNT, having over 35% sequence homology with BoNTs, and sharing much of the structural and functional features of BoNT, but is not a food poison due to the lack of NAPs. NAPs from one serotype of BoNT bind and protect another serotype of BoNT, thus these can also bind, protect and deliver vaccines, specially TeNT or TeNT vaccine due to sequence homology and common structural and functional domains. Therefore, the present invention uses BoNT's NAPs for oral and intranasal delivery of vaccines, specially tetanus vaccine.
Botulinum and tetanus neurotoxins have several common features including over 35% sequence homology, and antagonistic effects of heterologous protein fragments. Both the neurotoxins have similar secondary structural contents, and both have their ‘catalytic domain’ light chain on the C-terminal of their amino acid sequence. Tetanus is capable of causing botulism symptoms at high concentrations and blocks the release of acetylcholine from the presynaptic membranes just like the BoNT.
Similar mechanism of action for botulinum and tetanus neurotoxins at the molecular level occurs. Experimental studies reveal many similarities between the two neurotoxins including the membrane channel formation by the N-terminal fragments of their respective heavy chains and blockage of the neurotransmitter release from cultured cells and proteolytic activity against synaptobrevin-2. It is believed that there is enough common structural and functional similarity between BoNT and TeNT. The botulinum NAPs bind and protect TeNT similar to BoNT/A, providing basis for NAPs use an oral carrier of vaccine, specially tetanus vaccine.
BoNT is a food poison produced in the form of a complex with NAPs (see FIG. 1 ). NAPs protect BoNT from the low pH and proteases of the gastro-intestinal tract (Mahmut et al., 2002). NAPs are known to help translocate the BoNT across the mucosal layer of intestine as well as the nasal passage (Fujinaga et al., 2004). Given the similarity of structure of BoNT and TeNT and their common mode of action, Hn-33 is likely to bind and protect and translocate a tetanus vaccine across the mucosal layer (Mahmut et al., 2002).
Due to 35% sequence homology, and antagonistic effects of heterologous protein fragments, there is evidence for strong common structural features. Also, the ability of tetanus for causing botulism symptoms at high concentrations just like the BoNT it is likely that these proteins have a similar mechanism of action for botulinum and tetanus neurotoxins at the molecular level.
Experimental studies revealed many similarities between the two neurotoxins including the membrane channel formation by the N-terminal fragments of their respective heavy chains and blockage of the neurotransmitter release from cultured cells and proteolytic activity against synaptobrevin-2. Therefore there is enough common structural and functional similarity between BoNT and TeNT that NAPs of BoNT/A NAPs will bind and protect TeNT similar to BoNT/A, which provides basis for NAPs use an oral carrier of vaccines, especially tetanus vaccine.
The Tetanus toxoid vaccine, a second generation vaccine is prepared based on the non-toxic protein domain of TeNT, such as the heavy (H) chain for demonstrating its binding and delivery by NAPs.
The first steps include the preparation of a second generation tetanus vaccine based on the non-toxic heavy chain of the TeNT. FIG. 2 shows the schematic genomic map of pET32a, showing the location of TeNT heavy chain TeNT heavy chain plasmids were transferred to BL21(DE3) competent cells, and grown in the LB media. The E. coli culture was induced at OD600=0.5 by adding 0.2 mM IPTG and after induction culture continually grew at 16° C. for about 16 hours.
Second step is the preparation of NAPs from type A Clostridium botulinum
Finally, demonstration of NAPs binding to the second generation tetanus vaccine.
Present invention also provides method of preparation of the clone of DrTeNT as the third generation tetanus vaccine.
The third generation tetanus vaccine is prepared from and/or detoxified recombinant TeNT (DrTeNT) is cloned in pET32a vector between BamHI and HindIII sites with the N-terminal his-tag.
We also demonstrate protection of vaccine by NAPs against low pH and gastric protease, and further demonstration of the oral and intranasal delivery of the tetanus vaccine in rabbits (FIG. 12 ) by evaluating protection against challenge with TeNT, and allows one to compare the results with those obtained by intramuscular injections. Next, in this preferred embodiment, one may develop primate model for testing the effectiveness of the oral and intranasal delivery of tetanus vaccine by NAPs as an initial step towards testing this needle free delivery system in humans.
Preparation of NAPs from Botulinum Neurotoxin
Botulinum neurotoxin and purification of NAPs is done as per known method in the art (Kukreja et al., Toxicon, 2009 (53) 616-624).
Preparation of Tetanus Toxoid Vaccine
In this preferred embodiment, the cell paste culture suspended in about 1 L, 25 mM phosphate buffer, pH 7.4, containing 200 mM sodium chloride and 1% of TRITON®-X-100 added in the buffer (called basic buffer). After sonication, the cell lysate was centrifuged at 12,000 rpm for about 1 hour by using Thermo Scientific® Sorvall® Legend® RT Centrifuge and FIBERLite® F15-8x50C rotor, and then the supernatant is poured into a clean tube. The extract is thus obtained and is loaded to the pre-equilibrated Ni-NTA column.
After loading to the Ni-NTA affinity column, 10 mM imidazole is added to the basic buffer as a washing step, then protein binding to the column is eluted step by step at 50 mM imidazole, 100 mM imidazole, 200 mM imidazole and 500 mM imidazole. Next, one should combine the pool which has less contaminated bands and concentrate the pool by using CENTRIPREP®-30. After concentration, measure the protein concentration by UV, then prepare to do further binding experiments with NAPs.
Purification results of TeNT heavy chain obtained in this embodiment are shown in FIG. 3 .
Preparation of Detoxified Recombinant TeNT
The detoxified recombinant TeNT (DrTeNT) was prepared by cloning the TeNT gene and mutating two active site glutamic amino acid residues, E234 and E271 each to alanine residues. Cloning of Dr TeNT involved three steps: (1) pBN3 vector was used for cloning TeTx Light chain. (2) Two active sites mutation, E234A and E271A, was done which corresponds to E224 and E262 of BoNT/A active sites. (3) HC portion of TeNT was fused to construct full length tetanus neurotoxin plasmid with two active sites mutation, E234A and E271A.
E. coli BL21 (DE3) was used for the protein expression vector. After induction there were a large band at 150 kDa position which was the size of full length recombinant tetanus neurotoxin. After Ni-NTA affinity column, about 5 mg/ml pure protein was obtained which was soluble and stable.
FIG. 3 shows the results of purification of recombinant TeNT heavy chain through Ni2+-affinity column chromatography. Lane1 is MW standards. Lane 2 is Ni-NTA (Nickel-Nitrilotriacetic acid) affinity column washing with 10 mM imidazole added in the basic buffer. Lanes 3-6 are elution with 50 mM imidazole concentration. Lane 7 is elution with 100 mM Imidazole. Lane 8 is elution with 200 mM imidazole.
Preparation of NAPs-Tetanus Heavy Chain Composition
In another preferred embodiment, tetanus heavy chain has N-terminal his-tag which can bind to Ni-NTA affinity column, but NAPs which is purified from botulinum neurotoxin complex cannot bind to the Ni-NTA affinity column, so pull down assay is used for analysis to demonstrate if heavy chain would bind with NAPs. If Heavy chain has interaction with NAPs, both HC and NAPs would remain bound to NTA column and will be released together from Ni-NTA affinity column after eluting with imidazole.
Approximately equal molar ratio of tetanus heavy chain and NAPs are mixed together at pH 5.8. Binding reaction is carried out at room temperature for 2 hours. 25 mM phosphate buffer, pH 5.8, containing 200 mM NaCl is used for procedure of pull down assay. After reaction, the reaction mixture is diluted to a final imidazole concentration lower than 5 mM. The reaction mixture is loaded to the Ni-NTA affinity column, and 10 mM imidazole is added to the basic buffer to wash column at 20× bed volume, then the protein is eluted stepwise with 50 mM, 100 mM, 200 mM, 500 mM imidazole. The eluents are examined with SDS-PAGE to check the purity and intactness of the TeNT heavy chain, as illustrated in FIG. 3 .
Tetanus heavy chain purified from different batches is used for the binding experiments. After HC and NAPs reaction at room temperature, reaction mixture is reloaded to the Ni-NTA affinity column, with 10 mM Imidazole added to the basic phosphate buffer for washing column. After then protein is eluted with 50 mM, 100 mM, 200 mM imidazole. From the gel, it was observed that Tetanus heavy chain and NAPs were eluted together at 50 mM imidazole concentration (FIG. 4 ).
FIG. 4 shows binding of recombinant tetanus heavy chain (HC) preparation with NAPs as analyzed on Ni2+-affinity column chromatography. Lane 1 is MW. Lane 2 is intact heavy chain protein. Lane 3 is intact NAPs. Lane 4 is elution from Ni-NTA affinity column with imidazole 50 mM after loading HC and NAPs reaction mixture to Ni-NTA affinity column, Lane 4 showed HC and NAPs came together at 50 mM imidazole concentration.
NAPs results shown in FIG. 4 , lane 4, demonstrating that the NAPs successfully bound to the tetanus heavy chain. This observation would suggest that the tetanus heavy chain could be carried through the digestive system when administered orally in combination with NAPs. The NAPs are also expected to deliver the tetanus heavy chain or whole tetanus toxin across the intestinal mucosal layer.
Binding between tetanus heavy chain and NAPs is therefore demonstrated. Prepared clone of detoxified recombinant TeNT or DrTeNT which is full length TeNT with only two active site amino acid residues mutated (FIG. 5 and FIG. 6 ) has been achieved. DrTeNT has been successfully cloned in a pBN3 vector between E.coRI and PstI sites with C-terminal his-tag. The schematic diagram of new vector construct, named pDrT vector, is shown in FIG. 7 .
FIG. 5 shows nucleotide sequence of DrTeNT (3951 bp, including stop codon) which encodes for the full length double mutant tetanus gene. The start codon is enclosed in a green box, which is the same start for TeNT light chain; the double mutation sites are enclosed in orange boxes, GAA (Glu) to GCA (ALA) mutation; the heavy chain start codon (TCA) is enclosed in black box; and the stop codon is enclosed in a red box following the six His tag.
FIG. 6 shows the amino acid sequence alignment of DrTeNT, reference (data from wildtype genomic DNA), and CN3911 from Genebank. The two red boxes show that two active sites which got mutated in DrTeNT from wild type. The black boxes show that amino acid sequence variation from GeneBank C. tetani strain.
FIG. 7 shows a schematic representation of pDrT vector map containing DrTeNT gene (3966 bp include 6-His tag). DrTeNT was cloned in pBN3 vector (3452 bp), between E.coRI and PstI sites. The new construction of pDrT vector is 7418 bp in length.
Expression of DrTeNT gene in E. coli has been achieved and better than 99% pure DrTeNT is purified on a Ni-NTA affinity column (FIG. 8 ).
Binding of NAPs and DrTeNT is demonstrated using a G-100 gel filtration column chromatography performed in 50 mM sodium phosphate buffer, pH 5.8, containing 200 mM NaCl, showing a single peak elution of the mixture containing all the NAPs and DrTeNT (FIG. 9 ). Binding of NAPs and DrTeNT is confirmed by SDS-PAGE gel (FIG. 10 ). A model of the DrTeNT and NAPs complex is shown in FIG. 11 .
In another preferred embodiment the binding of the NAPs to tetanus heavy chain or DrTeNT is done using high-performance liquid chromatography (HPLC). In this preferred embodiment both NAPs and HC (used as a control) run separately when passed through the size exclusion column. After mixing them together at low pH conditions for binding with each other, and the mixture will appear as a single elution band.
In another preferred embodiment the binding of the NAPs to tetanus heavy chain is done using enzyme-linked immunosorbent assay (ELISA). In this preferred embodiment the tetanus heavy chain is coated on the 96 well plate at various different concentration Incubation of NAPs labeled with Horseradish Peroxidase (HRP) will show concentration dependent binding as monitored by colorimetric method by adding HRP substrate.
In another preferred embodiment the binding of the NAPs to tetanus heavy chain or DrTeNT is done using a BIACORE® surface plasmon resonance. In this preferred embodiment binding is evaluated on a gold chip.
In another preferred embodiment the binding of the NAPs to tetanus heavy chain or DrTeNT is done using isothermal calorimetry. In this preferred embodiment titration of a given concentration of NAPs with varying concentrations of tetanus vaccine candidate will show changes in heat evolved, thus allowing calculation of binding constant.
In another preferred embodiment the binding of the NAPs to tetanus heavy chain or DrTeNT is done using Fluorescence anisotropy. In this preferred embodiment the tetanus vaccine candidate can be labelled with a fluorescent probe, and titrated with the NAPs by monitoring fluorescence anisotropy.
In another preferred embodiment the binding of the NAPs to tetanus heavy chain, DrTeNT, or any other fragment thereof is demonstrated with x-ray crystallography.
In another preferred embodiment the binding of the NAPs to tetanus heavy chain or DrTeNT is demonstrated with electron microscopy.
In another preferred embodiment the binding of the NAPs to tetanus heavy chain, DrTeNT, or any other fragment thereof is demonstrated with NMR spectroscopy.
In another preferred embodiment the binding of the NAPs to tetanus heavy chain, DrTeNT, or any other fragment thereof is demonstrated with molecular dynamic simulations.
In another preferred embodiment the binding of the NAPs to tetanus heavy chain, DrTeNT, or any other fragment thereof is demonstrated with circular dichroism spectroscopy.
In another preferred embodiment the binding of the NAPs to tetanus heavy chain, DrTeNT, or any other fragment thereof is demonstrated with FTIR spectroscopy.
Evaluation of the Oral Delivery of Tetanus Vaccine
The effectiveness of inducing an immune response with oral delivery as well as subcutaneous delivery is shown in FIG. 12 . The first bar Sub Q Mix is subcutaneous mixture of NAPs with DrTeNT, next bar is same material via oral root and then oral DrTeNT alone. Although subcutaneous is more effective to illicit a response it seem that via oral mixture of both articles are more effective than the DrTeNT alone.
In another preferred embodiment neurotoxin associated protein from botulinum neurotoxin complex used as a delivery system for a tetanus vaccine.
In another preferred embodiment a neurotoxin associated protein from botulinum neurotoxin used as a delivery system for a tetanus vaccine.
In another preferred embodiment the tetanus heavy chain is used.
In another preferred embodiment the tetanus heavy chain linked to a botulinum toxin fragment is used.
In another preferred embodiment DrTeNT linked to DrBoNT is used.
In another embodiment a neurotoxin associated protein from botulinum neurotoxin complex is used as delivery system to botulinum vaccine.
In another embodiment neurotoxin associated proteins from botulinum neurotoxin complex is used as delivery system to botulinum vaccine.
In another embodiment, DrBoNT or any other fragment thereof is used as a vaccine.
In another preferred embodiment the neurotoxin associated protein is obtained from Clostridium botulinum.
In another preferred embodiment the neurotoxin associated protein is obtained from recombinant protein expression system.
In another preferred embodiment the neurotoxin associated protein is obtained from Clostridium botulinum bind to tetanus heavy chain.
In another preferred embodiment the detoxified recombinant form of whole tetanus neurotoxin (DrTeNT) containing mutated light chain and native heavy chain is used as vaccine alone for oral and intranasal delivery.
In another preferred embodiment the detoxified form of whole tetanus neurotoxin containing mutated light chain and native heavy chain is used as vaccine in combination with the neurotoxin associated protein for oral and intranasal delivery.
In another preferred embodiment the DrTeNT is combined with diphtheria vaccine element as a fusion protein.
In another preferred embodiment the DrTeNT is combined with pertussis vaccine element as a fusion protein.
In another preferred embodiment the DrTeNT is combined with vaccine elements of diphtheria and pertussis vaccine elements as a fusion protein.
In another preferred embodiment the group of neurotoxin associated proteins (NAPs) is used as a delivery vehicle for tetanus vaccine delivery.
In another preferred embodiment the tetanus vaccine is heavy chain of tetanus.
In another preferred embodiment the tetanus vaccine is light chain of tetanus.
In another preferred embodiment the tetanus vaccine is any fragment of tetanus.
In another preferred embodiment the vaccine is made of a fusion with DrTeNT or any of its derived fragments with diphtheria vaccine element.
In another preferred embodiment the vaccine is made of a fusion with DrTeNT or any of its derived fragments with pertussis vaccine element.
In another preferred embodiment the vaccine is made of a fusion with DrTeNT or any of its derived fragments with pertussis and diphtheria vaccine elements.
In another preferred embodiment the drug is created by combining DrTeNT or any of its fragments with any other drug candidate.
Applicant has described applicant's preferred embodiments of this invention, however it will be understood that the broadest scope of this invention includes modifications such as use of other equipment and laboratory procedures. Such scope is limited only by the claims as read in connection with the specification. Other advantages of the present invention will be apparent to those skilled in the art from the descriptions and the claims.
Advantage of the Invention
Present invention gives a novel composition of neurotoxin associated protein and vaccine.
The neurotoxin associated protein helps in oral or nasal delivery of vaccine, especially tetanus vaccine with or without diphtheria and pertussis.
Further, present invention gives a novel process for the preparation of second and third generation of vaccine.
Present invention also provides a novel process for the preparation of composition of neurotoxin associated protein and vaccine for oral delivery or nasal delivery.
Present invention also provides a method for evaluation of the efficiency of the oral delivery of vaccine.

Claims (13)

What is claimed is:
1. A process for the preparation of a vaccine and neurotoxin associated proteins (NAPs) composition, comprising:
mixing an equimolar ratio of the vaccine and neurotoxin associated proteins (NAPs) into a mixture that is maintained at a pH of from 5.5 to 6.2,
wherein the vaccine comprises a detoxified recombinant protein(s) derived from one or more proteins associated with an infectious disease selected from the group consisting of tetanus, diphtheria, pertussis alone or in combination thereof;
wherein the neurotoxin associated proteins (NAPs) are derived from Clostridium botulinum or botulinum neurotoxin, and the NAPs become chemically or recombinantly linked or otherwise associated to the vaccine during the mixing;
maintaining the mixture at the pH in the range of from 5.5 to 6.2 using 25 mM phosphate buffer at a pH 5.8 containing 200 mM sodium chloride solution;
reacting the mixture with at least 10 mM imidazole solution at an ambient temperature for from 2 to 3 hours;
loading the mixture to a Nickel-Nitrilotriacetic acid (Ni-NTA) affinity column; and
eluting the column to get a pure vaccine and neurotoxin associated proteins (NAPs) composition.
2. The process of claim 1, wherein the vaccine comprises a detoxified recombinant tetanus neurotoxin (DrTeNT).
3. The process of claim 1, wherein the imidazole concentration is at least 50 mM.
4. The process of claim 1, wherein the temperature is in the range of 20-30° C.
5. The process of claim 1, wherein the mixture is maintained at a pH of 5.8.
6. The process of claim 1, wherein the vaccine is a tetanus vaccine that is chemically or recombinantly linked or otherwise associated to a detoxified botulinum neurotoxin (DrBoNT) or any other vehicle, or carrier molecule.
7. The process of claim 1, wherein the vaccine is a tetanus vaccine that is chemically or recombinantly linked or otherwise associated to a fragment of detoxified botulinum neurotoxin (DrBoNT).
8. The process of claim 1, wherein the vaccine is a tetanus vaccine that is chemically or recombinantly linked or otherwise associated to the neurotoxin associated proteins (NAPs) of Clostridium botulinum or botulinum neurotoxin during the mixing of the equimolar ratio of the tetanus vaccine and the neurotoxin associated proteins (NAPs).
9. The process of claim 1, wherein the vaccine is a tetanus vaccine that is chemically or recombinantly linked or otherwise associated to neurotoxin associated proteins (NAPs) of any other serotypes and subtypes B to G of Clostridium botulinum or botulinum neurotoxin during the mixing of the equimolar ratio of the tetanus vaccine and the neurotoxin associated proteins (NAPs).
10. The process of claim 9, wherein the vaccine comprises a detoxified recombinant tetanus neurotoxin (DrTeNT) that is combined with a pertussis vaccine element as a fusion protein.
11. The process of claim 9, wherein the vaccine comprises a detoxified recombinant tetanus neurotoxin (DrTeNT) that is combined with a vaccine elements of diphtheria and pertussis vaccine elements as a fusion protein.
12. The process of claim 9, wherein the tetanus vaccine comprises detoxified recombinant tetanus neurotoxin (DrTeNT) formulated to be administered orally, subcutaneously or intraperitoneally, either alone or in combination with a vaccine elements of diphtheria and pertussis vaccine as a fusion protein.
13. The process of claim 9, wherein the tetanus vaccine comprises a detoxified tetanus neurotoxin (DrTeNT), or one or more of its fragments that is conjugated covalently or non-covalently with a therapeutic drug comprising a therapeutic small molecule or a therapeutic biomolecules.
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